The invention relates to new forms of pro-urokinase useful for thrombolytic therapy.
Pro-urokinase (pro-UK) is a single-chain form of a serine protease precursor, which is activated by plasmin to form two-chain urokinase (UK). Both pro-UK and UK activate, or convert, the zymogen plasminogen to the active enzyme plasmin, which degrades a series of plasma proteins included in fibrin clots. Consequently, both pro-UK and UK have been used for the treatment of thromboembolism.
There are certain undesirable side effects which can be caused by such treatment. Both pro-UK and UK can cause non-specific plasminogen activation, which leads to the degradation of fibrin, fibrinogen (fibrinogenolysis), and certain parts of platelets and blood vessel walls, and hemorrhagic diathesis. Pro-UK is more selective, i.e., specific, in its plasminogen activation at low doses than UK, because it activates only fibrin-bound plasminogen, whereas UK activates any plasminogen. However, pro-UK's specificity at low doses can be lost when it is administered at the high doses required for thrombolytic efficacy.
Pro-UK has certain properties which make it resemble both an "inactive" zymogen and an "active" enzyme. On one hand, pro-UK's zymogenic properties include its inert behavior in plasma, its failure to form sodium dodecyl sulfate-stable inhibitor complexes, and its relative resistance to inhibition by diisopropylfluoro-phosphate (DFP) or Glu-Gly-Arg chloromethylketone, which are potent chemical inhibitors of UK. Pro-UK also has a 200-fold lower plasminogen activating activity than UK.
On the other hand, pro-UK's enzymatic properties include its measurable intrinsic activity against both synthetic substrates and plasminogen, which is 10.sup.4.0-4.3 -fold higher than other serine protease zymogens such as trypsinogen or chymotrypsin. Pro-UK also has a lower Michaelis constant (KM) than UK against plasminogen, and, in the presence of fibrin fragment E.sub.2, pro-UK has been shown to be fully active against plasminogen without being activated to the UK form.
In spite of its potential use as a thrombolytic agent, when it is given in therapeutic doses, pro-UK's high intrinsic enzymatic activity initiates the undesirable non-specific systemic plasminogen activation noted above. As a result, thrombolytic therapy with pro-UK can still be associated with deleterious side effects.